Endophytic bacillus San.XJ-2 and application thereof

Abstract

The invention discloses an endophytic bacillus San.XJ-2 and application thereof, and belongs to the technical field of microorganisms, the endophytic bacillus San.XJ-2 is preserved in China Center for Type Culture Collection in Wuhan University, Wuhan, China on October 23, 2024, and the preservation number of the endophytic bacillus San.XJ-2 is CCTCC M 20242303. The endophytic bacillus can effectively release indissolvable potassium such as potassium feldspar in soil and increase the content of available nitrogen, phosphorus and potassium in the soil, has high saline-alkaline tolerance, and can effectively promote growth of crops after being prepared into a microbial agent and applied to the soil.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an endophytic bacillus San.XJ-2 and an application thereof. Background Art

[0002] Potassium is an indispensable element for plant growth and development. The total potassium content in the soil is higher than the total ammonia and total phosphorus content, but the insoluble potassium composed of aluminosilicates such as potassium feldspar and mica accounts for 95%, so the potassium that can be directly absorbed and utilized by plants is very small. With the adjustment of the structure of the orange industry, the crop yield and planting area continue to increase, and the demand for potassium fertilizers has also increased. Usually, chemical fertilizers are applied to supplement the plant's demand for potassium, but long-term and large-scale application of chemical fertilizers may have an adverse effect on the soil and pose a threat to the environment. The development of potassium-solubilizing bacteria biological preparations to replace chemical potassium fertilizers is a favorable way to solve plant potassium deficiency.

[0003] Potassium-solubilizing bacteria (KSB) can convert insoluble potassium in the soil into effective potassium that can be directly absorbed and utilized by plants, thereby promoting plant growth. Studies have shown that potassium-solubilizing bacteria isolated from rapeseed can effectively increase the biomass and potassium content of ryegrass after inoculation into the rhizosphere soil of ryegrass. Inoculation with potassium-solubilizing bacteria increased the root length, leaf area and biomass of rice, enhanced rice photosynthesis, and promoted the growth of rice seedlings. In addition, potassium-solubilizing bacteria not only have a positive effect on potassium absorption, but also promote plant growth by secreting indole-3-acetic acid (IAA), producing siderophores, and dissolving phosphates.

[0004] Similar to potassium deficiency, soil salinization is also a major factor limiting crop yields. High concentrations of sodium chloride (NaCl) have significant effects on plant growth, including ion damage, osmotic potential imbalance, and oxidative stress responses, which damage the soil system, thereby reducing plant photosynthesis and root activity, leading to reduced crop yields. Studies have shown that exogenous potassium ions can alleviate the negative effects of salt stress on tobacco photosynthetic apparatus. Exogenous potassium supplementation can alleviate the damage caused by NaCl to leaves by alleviating chlorophyll degradation and promoting chlorophyll synthesis, upregulating the gene expression of key proteins in this process, regulating abscisic acid signals, calcium signals, phosphorylation processes, and antioxidant mechanisms. However, there have been no reports on cases of KSB alleviating plant salt stress, and studying the role of KSB in alleviating plant salt stress has high application value. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides an endophytic Bacillus San.XJ-2 and an application thereof. The endophytic Bacillus can effectively release insoluble potassium such as potassium feldspar in the soil, increase the content of effective nitrogen, phosphorus and potassium in the soil, and the endophytic Bacillus has strong salt-alkali resistance. After being prepared into a bacterial agent and applied to the soil, it can effectively promote the growth of crops.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] An endophytic Bacillus (Bacillus endophyticus) San.XJ-2, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on October 23, 2024, and its deposit number is: CCTCC M 20242303.

[0008] A bacterial suspension comprises the above-mentioned endophytic Bacillus San.XJ-2.

[0009] A microbial agent comprises the above-mentioned endophytic Bacillus San.XJ-2.

[0010] Furthermore, fulvic acid is also included.

[0011] Furthermore, the content of endophytic Bacillus San.XJ-2 was 1.0×10 9 -1.0×10 10 cfu / mL.

[0012] The preparation method of the above-mentioned microbial agent comprises the following steps:

[0013] The isolated endophytic Bacillus San.XJ-2 was activated and cultured, then inoculated into a fermentation medium and cultured at 180 rpm and 30°C for 5 days. The obtained bacterial solution was centrifuged at 5000 rpm for 5 minutes to obtain a bacterial precipitate, which was resuspended in sterile water, and 5% fulvic acid was added. The potassium-dissolving bacterial agent was obtained after mixing.

[0014] Furthermore, the fermentation medium includes: 10 g / L glucose, 5 g / L yeast powder, 1 g / L (NH4)2SO4, 10 g / L Na2HPO4·12H2O, 1 g / L MgSO4·7H2O, 0.5 g / L CaCO3 and 2 g / L potassium feldspar powder. The pH of the medium is adjusted to 7.2 and sterilized at 115°C for 20 min before use.

[0015] Furthermore, the use of endophytic Bacillus San.XJ-2, bacterial suspension or microbial agent in improving the nutrient content of soil.

[0016] Furthermore, the use of endophytic Bacillus San.XJ-2, bacterial suspension or microbial agent in improving crop growth.

[0017] A method for increasing crop yield, using the above-mentioned endophytic Bacillus San.XJ-2, bacterial suspension or bacterial agent to cultivate crops.

[0018] The beneficial effects produced by the present invention are:

[0019] 1. The present invention selects Bacillus endophyticus with the ability to degrade insoluble aluminosilicate inorganic minerals and salt tolerance from the soil in the Lop Nur Salt Lake area of ​​Xinjiang, and prepares a potassium-dissolving bacterial agent by fermentation and cultivation with Bacillus endophyticus as the strain. The salt-tolerant potassium-dissolving bacteria can effectively promote the conversion of insoluble potassium and phosphorus and other nutrient elements into soluble nutrients, increase the content of fast-acting nutrients in the soil, promote crop growth and development, and increase yield. It has extremely important significance and application value for giving full play to the ecological fertility of the soil and maintaining the balance of the agricultural ecological environment.

[0020] 2. The potassium-dissolving bacteria agent prepared by the present invention has no possibility of discharging pollutants into the environment, and can effectively replace chemical fertilizers, thereby achieving a real reduction in the amount of chemical fertilizers and increasing their efficiency. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments.

[0022] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0023] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0024] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0025] Example 1: Screening and identification of potassium-solubilizing bacteria

[0026] 1. Activation of soil microorganisms

[0027] Soil samples were collected from the Lop Nur Salt Lake area in Xinjiang. The soil was immersed in sterile water at a ratio of 1:20 to isolate microorganisms. The soil microorganisms were activated with NB culture medium and shaken at 160 rpm and 30°C for 16 h. The components of NB culture medium were: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L inorganic salt, and the solvent was water.

[0028] 2. Screening of potassium-solubilizing bacteria

[0029] The activated microorganisms were diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , potassium separation medium containing 2g / L potassium feldspar was used to isolate potassium-dissolving bacteria strains. A potassium-dissolving bacteria strain was isolated, and the maximum diameter of its hydrolysis zone reached 11.2mm after 4 days of cultivation.

[0030] 3. Identification of potassium-solubilizing bacteria.

[0031] (1) Colony morphological characteristics:

[0032] Shape: On nutrient broth (NB) medium, colonies of endophytic Bacillus are rod-shaped;

[0033] Color: The colony is milky white;

[0034] Transparency: The colony is opaque and has a rough surface;

[0035] Edge: The edge of the colony is smooth and neat;

[0036] (2) Physiological and biochemical characteristics:

[0037] 1) Culture temperature: 25-37°C, the optimum temperature is 30°C;

[0038] 2) Gram stain: positive;

[0039] 3) VP reaction: negative;

[0040] 4) Casein hydrolase test: negative;

[0041] 5) Gelatin hydrolysis test: negative.

[0042] (3) The total genomic DNA of the potassium-dissolving bacteria was extracted, and the bacterial 16S rDNA universal primers 27F (sequence: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (sequence: 5'-CTACGGCTACCTTGTTACGA-3') were used for PCR amplification and product recovery. The 16S rDNA sequence of the strain San.XJ-2 was obtained after sequencing the PCR product. The obtained 16S rDNA sequence was compared with the strain sequence in the NCBI 16S database. The results showed that the homology between the potassium-dissolving strain San.XJ-2 and the endophytic Bacillus (Bacillus endophyticus) reached 100%, and the strain San.XJ-2 was an endophytic Bacillus (Bacillus endophyticus).

[0043] Example 2: Optimization of culture conditions of strain San.XJ-2

[0044] The effects of two single-factor culture conditions on the growth of strain San.XJ-2 were investigated. Factor 1 was temperature. Five temperature gradients (20°C, 25°C, 30°C, 35°C, 40°C) were set, the pH value was set to 7.5, 180rpm, and the OD600 of the bacterial solution was measured after 16 hours of culture. Different temperature conditions had a great influence on the growth and reproduction of potassium-solubilizing bacteria San.XJ-2. The results are shown in Table 1.

[0045] The results showed that when the temperature was 30℃, the number of strains in the fermentation broth reached the maximum, and the OD600 value was 1.674. Studies have shown that temperature affects the growth and reproduction of strains by affecting the activity of enzymes in cells. Under suitable temperature conditions, cell metabolism is vigorous, which accelerates the growth and reproduction of strains; when the culture temperature is 20℃, the number of potassium-solubilizing bacteria San.XJ-2 is the least, and the OD600 is 1.152. At this time, the temperature is low, the activity of enzymes in bacteria decreases, and metabolism slows down. When the temperature is 30℃, the number of strains in the fermentation broth is the largest. The possible reason for the analysis is that the most suitable temperature for enzymes related to cell growth in the microorganism is around 30℃. When the temperature is higher than 30℃, some cells in the microorganism will die due to low tolerance to temperature, slowing down the growth and reproduction of the strain. Therefore, in the five groups of experiments, the most suitable growth temperature of potassium-solubilizing bacteria San.XJ-2 is 30℃.

[0046] The second factor is pH value. Five pH gradients (6.0, 6.5, 7.0, 7.5, 8.0) were set, 30℃, 180rpm, and the OD600 of the bacterial solution was measured after 16h of culture. The effects of different pH on the growth and reproduction of strain San.XJ-2 are shown in Table 2. As can be seen from the table, when the initial pH of the culture medium is 6.5, the number of strains in the fermentation broth is the largest, the growth is the best, and the OD600 value of the bacterial solution is 1.666. Within a certain pH range, the number of strains increases first and then decreases with the increase of the pH of the bacterial solution, and the concentration of the bacterial solution is the largest when the pH is 6.5; when the pH continues to increase, the number of strains in the fermentation broth continues to decrease, and when the pH rises to above 8.0, the number of strains decreases significantly. Therefore, the optimum pH of potassium-solubilizing bacteria San.XJ-2 is 6.5. In summary, the optimum culture temperature of potassium-solubilizing bacteria San.XJ-2 in the fermentation medium is 30℃, and the optimum pH is 6.5.

[0047] Table 1 Growth of potassium-solubilizing bacteria San.XJ-2 at different temperatures

[0048] temperature 20℃ 25℃ 30℃ 35℃ 40℃ OD600 1.152 1.307 1.674 1.519 1.388

[0049] Table 2 Growth of potassium-solubilizing bacteria San.XJ-2 at different pH

[0050] pH 6.0 6.5 7.0 7.5 8.0 OD600 1.539 1.724 1.659 1.664 1.216

[0051] Example 3: Determination of potassium dissolving ability of strain San.XJ-2

[0052] 100 mL of potassium-dissolving active culture medium containing 2 g of potassium feldspar powder was placed in a 250 mL conical flask, inoculated with the bacteria, and cultured in a shaking incubator at 28°C and 160 rpm for 7 days. The culture solution was centrifuged at 3000 rpm for 5 min to obtain the supernatant, and the soluble potassium content in the supernatant was determined by flame spectrophotometry. There were 3 parallels in each group. No bacteria was added to the blank control (CK) group, the reported Bacillus megaterium strain 19749 was added to the T1 group, and the strain San.XJ-2 was added to the T2 group.

[0053] The results showed that after inoculation with potassium-solubilizing bacteria San.XJ-2, the soluble potassium content in the fermentation supernatant was significantly higher than that in the T1 group and CK group, at 7.12 mg / L. The soluble potassium content in the supernatant of the T1 group was 6.67 mg / L, and the soluble potassium content in the supernatant of the CK group was 4.94 mg / L. After inoculation with potassium-solubilizing bacteria San.XJ-2, the soluble potassium content in the supernatant was 44.13% higher than that in the CK group.

[0054] Example 4: Determination of salt tolerance of strain San.XJ-2

[0055] The strain San.XJ-2 and strain 19749 were inoculated into 100 mL NB medium containing different NaCl contents (0 g / L, 5 g / L, 10 g / L, 20 g / L, 40 g / L), and cultured at 28°C and 160 rpm for 16 h. 200 μl of the culture solution was taken at 12 and 16 h, and its OD600 was measured using an ELISA reader. The results are shown in Tables 3 and 4.

[0056] The results showed that after inoculation with potassium-dissolving bacteria San.XJ-2, the bacterial solution concentration was the highest in the groups containing 5g / L and 10g / L NaCl, and the OD600 reached 0.875 and 0.817 after 16h, respectively, and the bacterial solution concentration was the lowest in the group containing 40g / L NaCl. This indicates that strain San.XJ-2 has salt tolerance when the NaCl content is in the range of 5-20g / L, and stops growing when the NaCl content reaches 40g / L. Similar to strain San.XJ-2, after inoculation with strain 19749, the bacterial solution concentration was the highest when the salt concentration was 5g / L-10g / L, indicating that strain 19749 has salt tolerance when the NaCl content is in the range of 5-20g / L, and stops growing when the NaCl content reaches 40g / L. However, the salt tolerance of strain 19749 is significantly weaker than that of San.XJ-2.

[0057] Table 3 Growth of potassium-solubilizing bacteria strain San.XJ-2 under different salt concentrations

[0058] Salt concentration 0g / L 5g / L 10g / L 20g / L 40g / L 12h 0.435 0.606 0.627 0.319 0.088 16h 0.574 0.875 0.817 0.326 0.095

[0059] Table 4 Growth of potassium-solubilizing bacteria strain 19749 under different salt concentrations

[0060] Salt concentration 0g / L 5g / L 10g / L 20g / L 40g / L 12h 0.421 0.581 0.508 0.215 0.074 16h 0.588 0.856 0.784 0.298 0.084

[0061] Example 5: Strain San.XJ-2 can promote the growth of barley plants.

[0062] The experiment was divided into two groups: group T1 (5 kg sterile soil per pot, 1 ml of live bacteria concentration of about 1.0×10 9 cfu / mL of strain 19749 suspension, containing 25 mg of fulvic acid, 1.5 g of urea and 0.5 g of diammonium phosphate), T2 group (5 kg of sterile soil per pot, 1 ml of live bacteria concentration of about 1.0 × 10 9 cfu / mL of the strain San.XJ-2 bacterial suspension, the bacterial suspension contains 25 mg of fulvic acid, and 1.5 g of urea and 0.5 g of diammonium phosphate are added) and CK group (5 kg of sterile soil per pot, 1 ml of sterile water is added for seed dressing, and the other components are the same as the experimental group), and 3 biological replicates are set for each group. The barley variety is the cultivated variety Baudin, and the soil culture experiment is adopted. After 6 weeks, sampling is carried out to measure physiological indicators. All treated crops are cultured to maturity at 25°C and 16 / 8h light / dark alternation, harvested and weighed, and the results are shown in Table 5;

[0063] From the results in Table 5, it can be seen that potassium-dissolving bacteria can significantly promote the growth of barley seedlings. Compared with the absence of bacteria, the plant height of barley seedlings increased by 4.19%, the potassium content increased by 24.04%, the chlorophyll content increased by 16.71%, and the net photosynthetic rate increased by 11.06% after adding San.XJ-2. After adding 19749, the plant height of barley seedlings increased by 3.93%, the potassium content increased by 17.90%, the chlorophyll content increased by 10.62%, and the net photosynthetic rate increased by 10.14%. It shows that potassium-dissolving bacteria can enhance the growth of barley seedlings by improving the absorption of potassium nutrients by barley seedlings and promoting photosynthesis, and the bacteria prepared by strain San.XJ-2 has a more significant growth-promoting effect on barley seedlings.

[0064] Table 5 Effects of potassium-dissolving bacteria on the growth of barley seedlings

[0065]

[0066]

[0067] Example 6: Strain San.XJ-2 can effectively increase the available content of nitrogen, phosphorus and potassium in the soil.

[0068] The test was set up with Example 5. Soil samples were collected after collecting barley seedling plant samples, and the soil samples were ground after air drying. The alkaline nitrogen, available phosphorus and available potassium content in the soil were determined by alkaline diffusion method, sodium bicarbonate extraction-molybdenum antimony anti-spectrophotometry, and flame spectrophotometer method, respectively. The results are shown in Table 6. The results show that potassium-solubilizing bacteria inoculants can increase the effective content of nitrogen, phosphorus and potassium in the soil. After adding potassium-solubilizing bacteria San.XJ-2 inoculants, compared with CK treatment, the alkaline nitrogen, available phosphorus content and available potassium content in the soil increased by 12.23mg / kg, 20.48mg / kg and 44.36mg / kg, respectively. After adding potassium-solubilizing bacteria 19749 inoculants, compared with CK treatment, the alkaline nitrogen, available phosphorus content and available potassium content in the soil increased by 9.30mg / kg, 15.96mg / kg and 26.97mg / kg, respectively. This shows that potassium-solubilizing bacteria inoculants can effectively increase the nutrient content in the soil, improve soil texture and increase crop yield, and the inoculant prepared by potassium-solubilizing bacteria San.XJ-2 has a stronger activation effect on the effective nutrients in the soil.

[0069] Table 6 Effects of potassium-dissolving bacteria on nutrient content in soil

[0070]

[0071] Example 7: Effect of strain San.XJ-2 on the growth of barley seedlings under salt stress

[0072] They are T1 (5 kg sterile soil per pot, 2 ml of live bacteria concentration is about 1.0×10 9 cfu / mL of 19749 bacterial suspension, containing 100 mg of fulvic acid, 0.3% NaCl, 1.5 g of urea and 0.5 g of diammonium phosphate), T2 (5 kg of sterile soil per pot, 2 ml of live bacteria concentration of about 1.0 × 10 9cfu / mL of San.XJ-2 bacterial suspension, containing 100 mg of fulvic acid, and 0.3% NaCl, 1.5 g urea and 0.5 g diammonium phosphate were added) and CK (5 kg sterile soil per pot, 2 ml sterile water, 0.3% NaCl, 1.5 g urea and 0.5 g diammonium phosphate were added). Barley seedlings were sampled after 6 weeks of growth and physiological indicators were measured. The results are shown in Table 7. The results show that compared with no bacterial agent, the addition of potassium-dissolving bacteria agent 19749 and San.XJ-2 can enhance the resistance of barley to salt stress. After adding the bacterial agent prepared by San.XJ-2, the plant height of barley seedlings increased by 36.20%, the potassium content increased by 54.56%, the SOD content increased by 25.00%, the POD content increased by 10.67%, and the MDA content decreased by 13.57%. The effect of adding 19749 prepared bacteria agent on plant height and SOD content of barley seedlings was similar to that of San.XJ-2, but the effect on increasing potassium content, POD content and MDA content was weaker than that of San.XJ-2 bacteria agent. In summary, the addition of potassium-dissolving bacteria agent can significantly enhance the resistance of barley to salt stress, and has a significant growth-promoting effect. It can be promoted and used in production. Among them, strain San.XJ-2 can better alleviate salt stress of barley.

[0073] Table 7 Effects of potassium-solubilizing bacteria on the growth of barley seedlings under salt stress

[0074]

Claims

1. An endophytic Bacillus (Bacillus endophyticus) San.XJ-2, characterized in that It was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on October 23, 2024, and its deposit number is: CCTCC M20242303.

2. A bacterial suspension, characterized in that: It includes the endophytic Bacillus San.XJ-2 described in claim 1.

3. A bacterial agent, characterized in that: It includes the endophytic Bacillus San.XJ-2 described in claim 1.

4. The bacterial agent according to claim 3, characterized in that Also includes fulvic acid.

5. The bacterial agent according to claim 3, characterized in that The content of the endophytic Bacillus San.XJ-2 is 1.0×10 9 -1.0×10 10 cfu / mL.

6. Use of the endophytic Bacillus San.XJ-2 according to claim 1, the bacterial suspension according to claim 2 or the bacterial agent according to claim 3 in improving the nutrient content of soil.

7. Use of the endophytic Bacillus San.XJ-2 according to claim 1, the bacterial suspension according to claim 2 or the bacterial agent according to claim 3 in improving crop growth.

8. A method for promoting crop growth, characterized in that: The endophytic Bacillus San.XJ-2 described in the claim, the bacterial suspension described in claim 2 or the bacterial agent described in claim 3 is used to cultivate crops.

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